High Expression Of Recombinant Coagulation Factor VII In Chinese Hamster Ovary Cells

As an important coagulation factor, activated coagulation factor VII(FVIIa) is mainly used to treat the bleeding of hemophilia A patients with antibody, which is caused by FVIII treatment. Currently, FVIIa becomes the most common medicine for both hemophilia A and B patients. Besides hemophilia patients, FVIIa is also used to treat the clinical bleeding, especially the bleeding of puerperants. Currently, commercially available medicine of recombinant factor VII(rFVII) is “NovoSevenRT”, which is only produced by Novo Nordisk Company. Import price of “NovoSevenRT” for Chinese market achieved approximately 900 milions yuan per year. In 2013, national committee of Chinese New Medicine Research appointed the bio-similar medicine development of rFVII(Fund No.: 2013ZX09102033) to our group and the cooperative team of Soochow University. The task of our group is constructing the rFVII expressing cells and producing the prodrug of rFVII. According to the rule of food and drug administration(FDA) and China food and drug administration(CFDA) in the production of recombinant proteins predrugs, rFVII was expressed in Chinese hamster ovary(CHO) cells in this study. The constructed CHO cells were suspended to obtain rFVII-producing cells. Moreover, rFVII expression was enhanced by fed-batch culture, and high-cell density strategy was established. Results were shown as followed:1. rFVII expressing CHO cell line was constructing by the optimization of transfection efficiency and the screening of signal peptides. Based on FVII antibody and secondary antibody Alexa Fluor 488, rFVII expressing cells were labled by fluorescent dye, and positive cells percentage was accurately detected by flow cytometry. Depending on this method, transfection conditions of CHO cells was optimized. The highest efficiency was over 85 % under the condition of voltage 400 V, plasmid 20 ?g for three times with salmon sperm DNA as carrier DNA. In this study, CHO cells were selected to express rFVII expressing cell according to the rule of FDA and CFDA. The rFVII encoding gene with various signal peptides(Ori, IgK, Ht, Gl, Mutant) was inserted into high-GC content plasmid pMH3. The constructed plasmid was transfected into CHO cells and high rFVII expression sub-clones with various signal peptides were screened. After comparing in suspension culture, the highest rFVII expression of 5.42 mg?L-1 was detected in sub-clone with IgK signal peptide. rFVII cell line with signal peptide had higher expression and transcription level comparing with native signal peptide of FVII.2. The rapid screening method of high expression subclone was established based on flow cytometry. The traditional subclone screening method has many disadvantages, such as long time consuming and relying on personal experience. In this study, a rapid method for high expression subclone screening was established based on MoFlo XDP flow cytometry. The sorting condition for transfected cells was 3 cells per well in 96-well microplate. After detecting by dot and western blot for two times, a high rFVII expressing CHO cell line was obtained. After comparing with CHO cells from traditional method, two cells had a similar rFVII expression and cell growth. Moreover, the purity of positive cells was almost 100%. Depending on the established screening method, the consuming time for screening decreased from 8 month to 2 month. Depending on this method, sub-clone screening efficiency was effectively enhanced.3. rFVII expression was enhanced by optimizing culture conditons in suspension culture. Effects of different inoculum densities and culture temperatures on rFVII expression were investigated. Results showed that rFVII concentrations were 7.49 mg?L-1 and 7.18 mg?L-1 obtained by 1.0×106 cells?mL-1 and 34 °C, respectively. Moreover, temperature of 37 °C was suitable for cell growth. Therefore, rFVII expression cells were cultured in the two-stage strategy, which was culturing cells at 37 °C for 3 days and then fed on day 3 with 34 °C to the end of fermentation. After comparing different commercial available SFM, the highest rFVII expression in 3 L bioreactor with two-stage strategy was 24.51 mg?L-1 using B001 +F001 SFM. Cell cycle of CHO cells in two-stage strategy was analyzed and the percentage of cells in G1/G0 phase exceeded 80 % after feeding with F001. G1/G0 phase cells were hardly enhanced by linoleic acid and sodium butyrate. In 5 L bioreactor fed-batch culture, cell density maintained a high level during day 2 to day 8, and r FVII expression level was enhanced to 46.3 mg?L-1in 5 L bioreactor.4. High efficient rFVII purification based on immunoaffinity chromatography. After enrichment of r FVII by Q ion exchange column, the yield of rFVII was only 47.31 %. The low yield of rFVII led to one-step purification by immunoaffinity column in this study. The antibodies of immunoaffinity column were 3G3, 9E8, 9F11 and 10E5, which were all supplied by the cooperative team of Soochow University. After comparing the yield and purity of rFVII, 3G3 column had better purification efficient with yield of 92.3 % and purity of 91.48 %. Meanwhile, elution condition of pH value and Na Cl concentration were investigated. Results showed that rFVII yield and purity after eluting by pH 3.5 were 89.47 % and 90.31 %, respectively. However, low yield of rFVII was determined in elution bydifferent concentration of NaCl concentration. Effects of diluted and concentrated culture broth were also investigated in this study. Results indicated that 5-time concentrated rFVII culture broth and elution condition of pH 3.5 lead to the yield of 89.6 % and purity of 92.58 % in 3G3 immunoaffinity column. The purified rFVII was further activated and obtained rFVIIa showed the biological activity.